Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, has a non-segmented, negative-sense, single-stranded RNA genome. Its eleven kb genome has five genes which encode five structural proteins of the virus: the nucleocapsid protein (N), which is required in stoichiometric amounts for encapsidation of the replicated RNA; the phosphoprotein (P), which is a cofactor of the RNA-dependent RNA polymerase (L); the matrix protein (M) and the attachment glycoprotein (G) (e.g., see Gallione et al., 1981 J. Virol., 39:529-535; Rose and Gallione, 1981, J. Virol., 39:519-528; U.S. Pat. No. 6,033,886; U.S. Pat. No. 6,168,943).
In general, VSV is not considered a human pathogen, and as such, pre-existing immunity to VSV is rare in the human population. Thus, the development of VSV derived vectors has been a focus in areas such as immunogenic compositions. (e.g., vaccines) and the delivery of genes encoding therapeutic proteins. For example, studies have established that VSV can serve as an effective vector for expressing influenza virus haemagglutinin protein (Roberts et al., 1999 J. Virol., 73:3723-3732), measles virus H protein (Schlereth et al., 2000 J. Virol., 74:4652-4657) and HIV-1 env and gag proteins (Rose et al., 2001 Cell, 106(5):539-49). Other characteristics of VSV that render it an attractive vector include: (a) the ability to replicate robustly in cell culture; (b) the inability to either integrate into host cell DNA or undergo genetic recombination; (c) the existence of multiple serotypes, allowing the possibility for prime-boost immunization strategies; (d) foreign genes of interest can be inserted into the VSV genome and expressed abundantly by the viral transcriptase; and (e) the development of a specialized system for the rescue of infectious virus from a cDNA copy of the virus genome (e.g., see U.S. Pat. No. 6,033,886; U.S. Pat. No. 6,168,943).
The production of VSV vectored immunogenic compositions generally includes infecting a suitable cell culture (host) with recombinant VSV, growing VSV in cell culture, harvesting the cell culture fluid at the appropriate time and purifying the VSV from the cell culture fluid. The use of VSV vectors, and immunogenic compositions thereof, in clinical applications will require VSV samples (or doses) of appropriate purity in order to comply with safety regulations of the various drug safety authorities around the world (e.g., the Food and Drug Administration (FDA), the European Medicines Agency (EMEA), the Canadian Health Products and Food Branch (HPFB), etc.).
However, it is typically difficult to separate VSV from the cell culture contaminants (e.g., cell culture impurity proteins and DNA) and obtain VSV of appropriate purity and yield using the currently available VSV purification processes (e.g., purification via sucrose gradient centrifugation). For example, using the currently available purification processes, there is typically an inverse relationship between the purity and recovery (percent yield) of VSV samples, thereby making it difficult to manufacture sufficient quantities of purified VSV. Additionally, in today's bioreactor-based processes, increased cell concentrations and longer culture times result in higher VSV titers, with concomitant increases in cell debris and concentrations of organic constituents in the bioreactor fluid, further complicating VSV purification processes.
Sucrose gradient ultracentrifugation has been the standard method for virus purification (including VSV purification) since 1964 (Yamada et al., 2003 BioTechniques, 34(5):1074-1078, 1080; Brown et al., 1967 J. Immun., 99(1):171-7; Robinson et al., 1965 Proc. Natl. Acad. Sci., USA, 54(1):137-44; Nishimura et al., 1964 Japan. J. Med. Sci. Biol., 17(6):295-305). However, as virus concentrations increase, concomitant increases in cell debris, host DNA and protein impurities also occur, which are very difficult to remove at higher concentrations via sucrose gradient ultracentrifugation. In addition, sucrose gradient ultracentrifugation is extremely costly to scale-up. Concentration and purification of VSV by polyethylene glycol (PEG) precipitation (McSharry et al., 1970 Virol., 40(3):745-6) has similar problems of high impurity levels.
Relatively high quality virus has been obtained via size exclusion chromatography (Transfiguracion et al., 2003 Human Gene Ther., 14(12):1139-1153; Vellekamp, et al., 2001 Human Gene Ther., 12(15):1923-36; Rabotti et al., 1971 Comptes Rendus des Seances de l'Academie des Sciences, Serie D: Sciences Naturelles, 272(2):343-6; Jacoli et al., 1968 Biochim. Biophys. Acta, Genl Subj., 165(2):99-302). However, due to process cost and operating difficulty, it is generally not feasible for large-scale virus production. Affinity chromatography, such as heparin (Zolotukhin et al., 1999 Gene Ther., 6(6):973-985), lectin (Kaarsnaes et al., 1983 J. Chromatog., 266:643-9; Kristiansen et al., 1976 Prot. Biol. Fluids, 23:663-5) and Matrex™ Cellufine™ sulfate (Downing et al., 1992 J. Virol. Meth., 38(2):215-228), has found some application in virus purification. Heparin and lectin are generally not preferred (or used) for cGMP virus production due to possible leaching problems, which would require additional tests prior to product release.
Affinity purification of virus using Matrex™ Cellufine™ sulfate is an unresolved issue, due to efficiency of virus purification, virus quality and column regeneration. For VSV purification, very large affinity columns are needed (e.g., 0.2 L Matrex™ Cellufine™ sulfate resin per liter of cell culture; Wyeth Vaccine unpublished results). Low virus yield was observed when purified via ion exchange chromatography, either alone, or in combination with other types of traditional chromatographic techniques used in virus purification (International Patent Publication No. WO2006/011580; Specht et al., 2004 Biotech. Bioeng., 88(4):465-173; Yamada et al., 2003, cited above; Vellekamp et al., 2001 cited above; Zolotukhin et al., 1999, cited above; (International Patent Publication No. WO1997/06243; Kaarsnaes et al., 1983, cited above).
Thus, there is a current and ongoing need in the art for purification processes which can generate VSV at an appropriate level of purity and recovery (yield).